Growth and fermentation characteristics, biomass composition, lipid characterization and metabolic profiling analysis of two different Schizochytrium sp. strains, the original strain and the industrial adaptive strain, were investigated in the fed-batch fermentation process. The final cell biomass, total lipids content and docosahexaenoic acid (DHA) content of the adaptive strain were much higher than that of the original strain, and the DHA productivity of the adaptive strain was 146.7 mg L-1h-1, which was the highest one in all the published value. The starch and carbohydrate contents of the biomass in the original strain were higher than those in the adaptive strain and the protein contents in the biomass of both strains were relatively low (less than 2%). The lipid characterization of the two different stains illustrated that the percentages of DHA in total lipids and each lipid class did not change much in the adaptive strain while the percentages of DHA in neutral lipids was much less than that in polar lipids in the original strain, and the contents of unsaponifiable matters in the lipid produced by the adaptive strain were distinctly less than that by the original strain. The metabolic distinction extensively existed between these two strains were revealed by the score plot of Principal Component Analysis (PCA). In addition, potential biomarkers responsible for discriminating different strains were identified as myo-inositol, histidine, alanine, asparagine, cysteine and oxalic acid. These findings provided new insights into the industrial strain screening and further improvement of DHA production by Schizochytrium sp.
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Keywordsï¼šSchizochytrium sp.; docosahexaenoic acid; lipid characterization; metabolic profiling analysis
Polyunsaturated fatty acids (PUFAs), the critical membrane components in most eukaryotes and the precursors of many lipid-derived signaling molecules, are important nutrients and have many benefits on humans (Metz et al. 2001). Docosahexaenoic acid (DHA; 22:6, n-3), a kind of long-chain PUFAs, had drawn increasing attention for its benefits to human health including both infants and adults (Lauritzen et al. 2001; Nordoy et al. 2001; Ratledge 2004; Sijtsma and de Swaaf 2004). The traditional source of DHA is fish oil. However, the typical fishy smell, unpleasant taste, poor oxidative stability, seasonal variation and marine pollution of fish oil limit its use as a food additive. Microalgae or marine fungi may be interesting alternatives for fish oil as they are thought to be the primary producers of ω-3 PUFAs in the marine food chain. Schizochytrium sp., a kind of marine thraustochytrid, has the capability of synthesizing significant amounts of total lipid rich in DHA (Yokochi et al. 1998). Previous researches on the production of DHA by Schizochytrium sp. had mainly focused on achieving high cell density and high DHA content (Chi et al. 2007 2009, Fan et al. 2001, Ganuza et al. 2008, Liang et al. 2010, Ren et al. 2009 2010, Unagul et al. 2007, Wu et al. 2005), studies focused on the lipid composition as well as the distribution of fatty acids in individual lipid class for the industrial production of DHA were not much. In the present study, the fermentation performance of two different Schizochytrium sp. strains, the origin strain and the industrial adaptive strain, were investigated in a 10-L bioreactor using fed-batch fermentation. In addition, the biomass composition and lipid characterization of the fermentation results of the two strains were also studied. These results will provide useful information for the downstream processing of commercialized production of DHA -rich microbial lipids.
Metabolomics, the latest addition of functional genomics tools, focused on the systematic analysis of cellular behaviour at molecular level and emerged as a powerful tool capable of screening a large number of metabolites in biological samples and providing valuable physiological information on numerous biological systems (Villas-Bôas et al. 2005, Baker 2011). The intracellular metabolome responds more rapidly to environmental changes than the transcriptome and proteome as the signaling can use existing receptors and enzymes to change fluxes within a rigid metabolic network (Raamsdonk et al. 2001). Up to now, research on the metabolomic profile analysis of the DHA producing strain Schizochytrium sp. had not been reported yet; little had been known about the metabolome features of this oleaginous microorganism systematically. In this paper, gas chromatography-mass spectrometry (GC-MS) was applied to detect the changes of intracellular metabolites during the fed-batch fermentations of the origin strain and the industrial adaptive strain. Principal components analysis (PCA) of intracellular metabolites was performed to distinguish the biomaekers during the fed-batch cultures of these two different strains. Interpreting the metabolomic distinction of the original and the industrial adaptive strains in fermentation processes would provide new insights into the industrial strain screening and optimization of this commercial DHA-producing microorganism.
Material and methods
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Schizochytrium sp. CCTCC M209059, which was isolated from seawater and stored in China Center for Type Culture Collection (CCTCC), was used in the present study as the original strain. This strain was preserved in 20% (v/v) glycerol at -80â„ƒ. The industrial adaptive strain was the industrialized strain which was screened and optimized in the laboratory of Jiangsu TianKai Biotechnology Co., Ltd.
The seed culture medium and the conditions were the same as those used in our previous study (Ren et al. 2010). The culture preserved in the glycerin tube was inoculated into a 250-mL flask with 50mL medium and cultivated for 24h. After three generations of cultivation, the seed culture (10%, v/v) was then transferred to a 10-L fermentor with a working volume of 7 L. The fermentation conditions were the same as our previous study (Qu et al. 2011).
Cell dry weight, total lipids, and fatty acid analysis
The measurements of cell dry weight, total lipids, individual fatty acid, glucose, glutamate and pH used the same method as our previous study (Qu et al. 2011).
Quantification of starch and protein
The starch content in Schizochytrium sp. was quantiï¬ed in duplicate using a modiï¬ed version of the method used by Davis et al. (2006).
Protein extraction was performed according to (Weis et al., 2002) with some modiï¬cations.
Analysis of lipid fractions
The total lipid (2.5g) was fractionated to neutral lipids (NLs) and polar lipids (PLs) by elution on a silica column, initially with petroleum ether/diethyl ether (9:1) and then with methanol. After evaporation of the eluate, the amount of each lipid fraction was determined gravimetrically. The lipid was fractionated by thin-layer chromatography (TLC) on a silica gel plate by developing with chloroform/acetone/methanol/acetic acid/water (50:20:10:10:5, v/v).
Isolation and analysis of unsaponifiable matters, β-carotene, squalene and cholesterol from lipid
Unsaponifiable matters were isolated from lipid by saponification (Dhara et al. 2010). The β-carotene content was analyzed according to Hart et al (1995) with some modifications. Dionex U3000 HPLC equipped with ultimate 3000 variable wavelength detector and reversed-phase Superspher C18 columns (150-4.6 mm i.d. 5μm, Venusil) at a column temperation of 30â„ƒ were used.
Squalene was analyzed by GC system (GC- 2010, Shimadzu, Japan). The GC was equipped with a capillary column (DB-23, 60m-0.22 mm) and flame ionization detector (FID).
The measurement of cholesterol was according to the GB/T 5009.128-2003.
Sampling, quenching, and extraction of intracellular metabolites
In order to compare the metabolome profiles of Schizochytrium sp. at different fermentation stages, cells were sampled every twelve hours during the fermentation period. Cells were quenched and extracted according to Ding et al (2009a, 2009b) with slight modifications.
Metabolome analysis by GC-MS
The sample was analyzed by GC-MS as described previously (Strelkov et al., 2004). The GC-MS system consisted of a Finnigan Trace gas chromatograph and a Trace mass spectrometer (Thermo Finnigan, San Jose, USA).
Multivariate statistical analysis
Principal components analysis (PCA) was carried out after preprocessed by mean-centering and scaling to analyze the dataset with STATISTICA (Kind et al., 2007).
Time courses of fed-batch fermentation of the original strain and adaptive strain (Growth and fermentation characteristics)
Growth and fermentation characteristics of the two different Schizochytrium sp. strains were investigated by the fed-batch cultivations, respectively. The results indicated that the fermentation performances of the two strains were distinctive. As shown in Fig.1, the adaptive strain exhibited much better performance in growth and glucose metabolism than the original strain, and the final cell biomass, total lipids content and DHA content of the adaptive strain were 67.21, 42.56 and 17.02 g L-1, much higher than that of the original strain (56.01, 25.82 and 8.19 g L-1). Furthermore, the DHA productivity of the adaptive strain was 146.7 mg L-1h-1, over two times of that of the original strain (60.2 mgL-1h-1). The observations described above indicated that the industrial adaptive strain had a great improvement in the DHA producing capability than the original strain.
Comparison of biomass composition and lipid characterization of the original and adaptive strain
The differences of the biochemical composition of the two different Schizochytrium sp. strains were investigated in fed-batch culture. Specific concentrations of starch, carbohydrates, proteins, and lipids in the original and adaptive strains were shown in Table 1. For the industrial adaptive strain, lipid was the quantitatively most important cell constituent, making up over 60% of the biomass, much more than the original strain (about 46% of the biomass). The starch and carbohydrate contents of the biomass in the original strain were 49.1 and 135.1 mg g-1, much higher than those in the adaptive strain (29.7 and 124.2 mg g-1). The protein contents in the biomass of both strains were relatively low (less than 2%).
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Lipid fractions of the two different Schizochytrium sp. strains were analyzed by elution on a silica column. As shown in Figure 2, the neutral lipids made up over 85% of the total lipids in both strains. The difference in the lipids fractions between the two different strains was not significant. The polar lipids content in the original strain was 13.09%, a little more than that in the adaptive strain (11.32%). The fatty acids composition of total lipids and individual lipid class of the two strains was further analyzed and the results were listed in Table 2. In the adaptive strain, the percentages of DHA in total lipids and each lipid class did not change much (45.53% in total lipids, 45.47% in neutral lipids and 44.12% in polar lipids). However, in the original strain, the percentages of DHA in total lipids and each lipid class change much among different lipid class (41.45% in total lipids, 35.51% in neutral lipids and 52.01% in polar lipids). In addition, the ratios of the total unsaturated fatty acids divided total saturated fatty acids in the adaptive strain were obviously higher than that in the original strain, indicating that the fluidity of the lipid produced by the adaptive strain was much better than that produced by the original strain.
Three main unsaponifiable matters, β-carotene, squalene and cholesterol, were isolated from lipid by saponification and analyzed by HPLC and GC-MS, respectively. Table 3 illustrated that the contents of the main unsaponifiable matters in the lipid produced by the adaptive strain were distinctly less than that by the original strain. In the adaptive strain, the contents of β-carotene, squalene and cholesterol were 34.91μg g-1, 19.99 and 40.93 mg g-1 while in the original strain, these contents were 50.28μg g-1, 26.58 and 49.59 mg g-1.
Metabolic profiling and Multivariate statistical analysis of the fed-batch cultures of the adaptive strain and the original strain
The metabolites of the adaptive Schizochytrium sp. strain and the original Schizochytrium sp. strain were analyzed by GC-MS to compare their metabolic differeneces in the fed-batch cultures. More than 60 putative intracellular metabolites were detected and 39 of them were identified and quantified in all samples at different fermentation stages. The identified metabolites included a range of intermediates from central carbon metabolism, such as amino acids, organic acids, alcohols as well as sugars.
Unsupervised evaluation and independent t-test were used to observe global trends that were remarkably different between the two different strains, providing comparative interpretation of the metabolic changes for the adaptive Schizochytrium sp. strain. The metabolic profile dataset was statistically analyzed by PCA to gain insight into the nature of the multivariate data and evaluate biological alteration (Fig. 3a and 3b). PCA score plot (Figs. 3b) demonstrated that the maximum variability in the data set clearly differentiated between different strains (precisely, between the adaptive strains and the original strain), with the first component (PC1) covering 46.45% of the data variance and the second principal component (PC2) explained 16.15% of the total detected metabolites pools variance.
PCA loading plot (Fig. 3a) showed the potential biomarkers for distinguishing samples from the two different Schizochytrium sp. strains. The potential biomarkers which were significant for distinguishing the two different strains mainly included myo-inositol, histidine, sulfurous acid, eicosane, alanine, asparagines, cysteine and oxalic acid.
Besides the multivariate data analysis to examine the global variations between different experimental conditions, the variances of individual compounds were analyzed in greater detail. The differences of the content of several intracellular metabolites, which were the potential biomarkers analyzed by the PCA loading plots, myo-inositol, histidine, alanine, asparagine, cysteine and oxalic acid were measured and the results were showed in Fig 4. It is illustrated that the content of myo-inositol in the adaptive strain was higher than that in the original strain during the early stage of the fermentation, but in the late stage the situation was the reverse. The trends of the contents of the four amino acids, histidine, alanine, asparagines and cysteine were similar that the adaptive strain accumulated lower contents of these amino acids than the original strain during the whole fermentation process. In addition, the content of oxalic acid in the adaptive strain was lower than the original during the whole culture period, the same trend as the amino acids.
In the present study, fermentation performance, biomass composition, lipid characterization of two different Schizochytrium strains, the original strain and the industrial adaptive strain, were investigated in the fed-batch fermentation culture to study the difference of the industrial fermemtation potential of the two strains. The metabolic profiling analysis of the two strains was also investigated to study the metabolism difference of the two strains to understand the adaptive mechanism of the industrial strain screening and optimization of Schizochytrium sp.
It could be seen from the fermentation results that the industrial adaptive strain revealed much better performance in producing total lipids and DHA than the original strain. The DHA productivity of the adaptive strain was 146.7 mgL-1h-1, which exceeded the highest published value of 134 mgL-1h-1 by Schizochytrium sp. SR21 (Yaguchi et al. 1997), 115 mgL-1h-1 for Schizochytrium mangrovei Sk-02 (Unagul et al. 2006), 117 mgL-1h-1 for strain 12B (Perveen et al. 2006), 123 mgL-1h-1 for Aurantiochytrium limacinum SR21 (Huang et al. 2012) and 93 mgL-1h-1 by using Aurantiochytrium sp. T66 (Jakobsen et al. 2008). These indicated that the industrial adaptive strain had the commercialization potential of producing DHA-rich single cell lipids.
The biomass compositions of the two different Schizochytrium strains were also investigated in fed-batch cultivation. The industrial adaptive strain had more lipid constituent and less starch and carbohydrate in its biomass than the original strain. These results were consistent with the fermentation performances of the two strains. The biochemical composition in this study was different from the reported biomass composition of another DHA-producing microorganism Crypthecodinium cohnii CCMP 316 (Pleissner et al. 2012). In that stain, starch made up 50% of the biomass, lipids and proteins each made up 12-15% of the biomass. It was also an explanation of the increasing interests of the research of the microorganism Schizochytrium in DHA production other than Crypthecodinium cohnii. The biomass composition of the two strains provided useful information for the downstream processes such as lipid extraction and biomass recycling.
Fig 2 illustrated that the neutral lipids made up about 85% of the total lipids in both strains and the polar lipids content in the adaptive strain was less than the original strain. The lipid class distribution of the total lipids in both strains in our research was close to those in the literature. Schizochytrium limacinum was reported to have about 82% of neutral lipids and 14% of polar lipids (Wang et al. 2012), and the lipids in Schizochytrium mangrovei FB3 contained over 90% of neutral lipids and 5% of polar lipids (Fan et al. 2007). In the adaptive strain, the percentages of DHA in total lipids and each lipid class did not change much, while in the original strain, the percentages of DHA in neutral lipids was much less than that in polar lipids. As the polar lipids were mainly consisted of phospholipids, the essential components of cell membranes, and these polar lipids would be removed from the extracted lipids during the downstream processes. Thus, during the downstream processes, the extracted lipids from the original strain would lose more DHA than the adaptive strain. Additionally, the higher content of the total unsaturated fatty acids in the adaptive strain demonstrated that the fluidity of the lipid produced by the adaptive strain was much better than that produced by the original strain. Furthermore, the contents of three main unsaponifiable matters in the lipid produced by the adaptive strain were distinctly less than that by the original strain, and the exits of these unsaponifiable matters would increase the difficulty of oil refining.
By the analysis of the fermentation characteristics, biomass composition, lipid characterization of the two strains, the industrial adaptive strain showed much better DHA productivity as well as the better lipid quality and less processing cost of the final oils. The analysis of the biomass composition and lipid characterization also provided important information for the downstream process of future commercialized production of DHA -rich microbial lipids.
In the present study, the metabolic difference between the adaptive strain and the original strain of Schizochytrium sp. were studied by metabolic profiling. Clear differentiation by PCA score plot (Fig. 3b) showed that significantly metabolic distinction extensively existed between the adaptive and the original strain during fed-batch fermentation. Throughout the industrial fermentation process, Schizochytrium sp. was subjected to a variety of environmental stresses, including osmotic pressure, toxic metabolites accumulation and gradual nutritional depletion. Thus, the strain needed to adjust its metabolism to the industrial fermentation conditions.
PCA loading plot (Fig. 3a) revealed the potential biomarkers that were significant for distinguishing the two different strains. The differences of the content of several potential biomarkers, myo-inositol, histidine, alanine, asparagine, cysteine and oxalic acid were also compared. These results would contribute to understand the adaptive features of Schizochytrium sp. strain in the industrial fed-batch fermentation process. It could be seen from Fig.4 that the adaptive strain accumulated higher concentration of intracellular myo-inositol than the original strain during the early stage of the fermentation, but the original strain had a much higher content of myo-inositol in the late stage. Myo-inositol was a kind of poyolsl, and polyols was reported to function as carbohydrate reserves compounds, and played important roles in osmoregulation, storage of reducing power and coenzyme regulation in living organisms (Jennings 1984). It was reported that polyols accumulated in P. chrysogenum in response to salinity (Adler et al. 1982). Myo-inositol was a precursor of phosphoinositol (PI), which was important for regulation of membrane trafficking and several nuclear functions of cells (York 2006). It had been found that myo-inositol induced positive effects on wine yeast under concomitant thermal and osmotic stress (Caridi 2002). Furukawa et al. (2004) also reported that intracellular myo-inositol content of S. cerevisiae was one of the important factors which contributed to high ethanol tolerance. One research on ethanol stress response of diploid and haploid yeast by transcriptomics also had the similar results that two myo-inositol synthesis related genes, INO1 and INM1, were dramatically induced by ethanol stress, especially in a-type haploid (Li et al., 2010a). So in this study, higher level of myo-inositol accumulated in the adaptive strain during the early stage of fermentation could be due to its faster metabolic adjustment than the original strain to adapt to the fermentation environment.
The contents of the four amino acids, histidine, alanine, asparagine and cysteine, had the same trend that they were higher in the original strain than the adaptive strain during the whole fermentation course. It suggested that these metabolites were critical in cellular defense against the stress of fermentation environment, and the adaptive strain had been adapted to the combined stresses in the amino acids metabolism. It was found that amino acids accumulated as a general stress response under cold, heat and oxidative stresses (Jozefczuk et al. 2010). In addition, the increased amino acid levels of Schizochytrium in the original strain could be, at least in part, a result of increased protein degradation (Mandelstam 1963; Jozefczuk et al. 2010). The degradation of proteins could partly be due to the need to eliminate abnormal proteins formed as a result of stress, and it could be explained that the protein degradation was to increase the availability of amino acids required for the synthesis of new proteins which were important for survival under unfavorable condition (Willetts 1967). Furthermore, in our results, higher levels of amino acids such as alanine, asparagine and cysteine in the original strain during the fermentation process revealed that nitrogen metabolism was more active in the original strain than in the adaptive strain. Amino acids were critical parts of carbon and nitrogen metabolism, and precursors of a wide range of cell components including proteins, nucleotides, and other nitrogen-containing compounds. Analysis on amino acids provided insights in metabolic coordination, as well as the relationship between carbon-nitrogen status and amino acid metabolism (Fritz et al., 2006). Additionally, higher content of amino acids in the original strain also reflected a decrease of the TCA cycle flux since these metabolites were formed either during glycolytic pathway or from its intermediate branches. In the original strain, higher levels of valine and alanine, which were derived from pyruvic acid, could account for the better metabolic activity around the pyruvic acid branch point. Asparagine functioned in transport and storage of nitrogen in some species (Zulak et al., 2008). Higher levels of asparagine in the original strain than the adaptive strain revealed that nitrogen transport and storage were more demanded in the original strain during the fed-batch fermentation process.
The content of oxalic acid in the adaptive strain was also lower than the original during the whole culture period. Oxalic acid probably originated from hydrolysis of oxaloacetate (Kubicek et al., 1988), which was an intermediate of the TCA cycle. Thus, the higher level of oxalic acid in the original strain than the adaptive strain revealed a decrease of the TCA cycle flux, and this speculation was in accordance with the deduction that mentioned above about the content difference of amino acids in the two strains.
The strategy of GC-MS-based metabolomics in this study provided general metabolic profiles of two different Schizochytrium sp. strains during the fed-batch processes. The observed variations of intracellular metabolites led to a better understanding of different metabolic status of the adaptive and the original strain during fermentation course. Understanding the mechanisms involved in the adaption of Schizochytrium sp. strain to the industrial fermentation conditions in combination with target metabolic engineering appeared to be a very promising strategy to obtain more efficient strains that could be used for efficient DHA producing processes.
This work was financially supported by the National Basic Research Program of China (no. 2011CBA00802), the Scientific Research Project for Post-graduate in Jiangsu Province (no. CXLX11_0366), the Natural Science Foundation of Jiangsu Province (no. BK2012424), the National Science and Technology Pillar Program (no. 2011BAD23B03), and the National High Technology Research and Development Program of China (no. SS2012AA021704).